DESCRIPTION: The long term objective of this project is to provide a complete kinetic and thermodynamic description ("model") of the sodium pump or Na,K-ATPase. The sodium pump is an integral protein of the plasma membrane of nearly all animal cells and is responsible for maintenance of the transmembrane Na and K gradients that underlie the generation of resting and action potentials, cell volume and pH regulation, fluid secretion and reabsorption, secondary transport of nutrients and other ions, etc. The action of digitalis drugs on the heart is almost certainly mediated through the sodium pump. The sodium pump in humans consumes an appreciable fraction of all ATP made in the body. The proposal comprises four specific aims: (1) To continue to analyze steady-state kinetic and thermodynamic relationships between modes of operation (forward Na/K; backward K/Na; Na/Na exchange; K/K exchange) of the pump with special attention to the effects of cell membrane potential. (2) To continue to analyze pre-steady-state voltage jump-induced current transients in the Na/Na exchange (and possibly K/K exchange) mode with the purpose of establishing precursor-product relationships between the 3 resolvable components of the transients. (3) To generate and analyze [ATP] jump-induced pump current transients under voltage clamp. These three aims will be pursued on internally dialyzed giant axons of Loligo pealei which allow (i) control of ionic and biochemical conditions and membrane potential, and (ii) measurement of isotopic fluxes and (minuscule) transmembrane currents, from hour-long DC stability to microsecond resolution. Kinetic analysis will be done by rigorous least-squares fitting with attention to internal consistency between (i) certain parameters shared by the various modes and (ii) steady-state and pre-steady-state parameters describing a given reaction. Of great interest is the effect of cell membrane potential on kinetic parameters (i.e. rate and equilibrium constants; apparent affinities; Hill coefficients) because these effects reveal important physical and structural constraints such as the nature and depth of access wells or the amount of charge tranversing a field during a given step. The final specific aim is (4) to use Xenopus oocyte expression (i) to identify on Xenopus alpha subunit clones the residues that interact with the lactone ring of cardiotonic steroids and (ii) to carry out initial characterization of the cloned squid pump alpha subunit and examine a small number of putative mRNA editing sites for possible effects on macroscopic pump characteristics.